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Biomedical subjects

E Benoit

Publications and source records attributed to E Benoit.

At least 19 recordsLinked to original sources
PubMed2006

Oxaliplatin, an anticancer agent that affects both Na+ and K+ channels in frog peripheral myelinated axons.

The use of oxaliplatin, a relatively new chemotherapeutic agent, is somewhat limited since it produces a specific peripheral neuropathy regarding other neurotoxic anticancer platinum analogues. In order to investigate the mechanism of such a peripheral neuropathy, the effects of 1-100 micromol/l oxaliplatin were assessed on the nodal ionic currents of single frog myelinated axons as a model of peripheral excitable membranes. Oxaliplatin decreased both Na(+) and K(+) currents in a dose-dependent manner and within 5-10 min, without producing any marked changes in the current kinetics. It was about three to eight times more effective in reducing the Na(+) than the K(+) current. In addition, it shifted the voltage-dependence of both Na(+) and K(+) conductances towards negative membrane potentials. A negative shift in the steady-state inactivation-voltage curve of the peak Na(+) current was also observed in the presence of oxaliplatin. These effects were not reversed by washing the myelinated axons with an oxaliplatin-free solution for at least 30 min. It is concluded that oxaliplatin modifies the voltage-dependent ionic channels mainly by altering the external surface membrane potential. The knowledge of such a mechanism may help to counteract the neurotoxic action of this anticancer agent.

Animals↗
PubMed2005

Hypocalcaemia due to nutritional calcium deficiency and hypoparathyroidism in an adult dog.

A 13-year-old intact male poodle had suffered periodic tetanic crises for two months. It was cachectic and moderately dehydrated, and during the crises blindness, a stiff gait and behavioural changes were observed. Routine haematological and biochemical profiles showed that it was severely hypocalcaemic, with a corrected plasma calcium concentration of 1.13 mmol/litre (reference range 2.25 to 3 mmol/litre). The dog was fed a home-made diet composed of chicken and basmati rice cooked with a soup bouillon cube; an analysis of its daily allowance indicated that the dog was generally malnourished and received only 0.222 g of calcium per day rather than the 0.6 g it required. In addition, the dog had a low blood concentration of parathyroid hormone of 12 ng/litre (reference range 20 to 80 ng/litre). Supplementing the dog with calcitriol for four days and correcting its diet increased its blood calcium to the lower part of the reference range and resolved the clinical signs, although its parathyroid hormone concentration was still low one year later.

Animals↗
PubMed2004

CEACAM6 is a determinant of pancreatic adenocarcinoma cellular invasiveness.

Pancreatic adenocarcinoma is among the most aggressively invasive malignancies. The immunoglobulin superfamily member carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers. We sought to define the role of CEACAM6 in pancreatic adenocarcinoma cellular invasiveness. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express low levels of CEACAM6. Retrovirally mediated RNA interference was used to silence CEACAM6 expression in BxPC3 cells, which inherently overexpress CEACAM6. Cellular invasiveness was quantified using a modified Boyden chamber assay. Overexpression of CEACAM6 increased Capan2 cellular invasiveness, whereas CEACAM6 knockdown attenuated BxPC3 invasiveness. A role for the c-Src tyrosine kinase in mediating CEACAM6-dependent invasiveness was defined using constitutively active and dominant-negative c-Src expression constructs. c-Src-dependent modulation of matrix metalloproteinase-9 activity contributes significantly to the increased cellular invasiveness induced by CEACAM6 overexpression. Levels of CEACAM6 expression can modulate pancreatic adenocarcinoma cellular invasiveness in a c-Src-dependent manner. This pathway warrants further investigation as a target for therapy.

Adenocarcinoma↗
PubMed2001

Cloning, sequencing, tissue distribution, and heterologous expression of rat flavin-containing monooxygenase 3.

The sequence of rat FMO3 was obtained by RT-PCR and 5'/3' terminal extension. Complete cDNA was amplified, cloned, and sequenced. The cDNA encodes a protein of 531 amino acids which contains the NADPH- and FAD-binding sites and a hydrophobic carboxyl terminus characteristic of FMOs. This sequence is 81, 81, and 91% identical to sequences of human, rabbit, and mouse FMO3, respectively, and 60% identical to rat FMO1. Rat FMO3 was expressed in Escherichia coli. The recombinant protein and the native protein purified from rat liver microsomes migrated with the same mobility (56 kDa) as determined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Recombinant rat FMO3 showed activities of methimazole S-oxidation, and NADPH oxidation associated with the N- or S-oxidation of trimethylamine and thioacetamide, in good concordance with those reported for human FMO3. When probed with rat FMO3 cDNA (bases 201 to 768), a strong signal corresponding to the 2.3-kb FMO3 transcript was detected in RNA samples from rat liver and kidney while a weak signal was observed with lung RNA samples. In contrast, the probe did not hybridize with any RNA from brain, adipose tissue, or muscle.

Amino Acid Sequence↗
PubMed2001

The scorpion alpha-like toxin Lqh III specifically alters sodium channel inactivation in frog myelinated axons.

The effects of 1-100 nM Lqh III, an alpha-like toxin isolated from the scorpion Leiurus quinquestriatus hebraeus, were assessed on the nodal membrane potential and ionic currents of single frog myelinated axons. In current-clamped axons, Lqh III increased the duration of action potentials without markedly affecting the peak amplitude and the resting membrane potential. The toxin was less effective when the resting membrane potential of axons was increasingly more positive. The Lqh III-induced increase in action potential duration was not due to the blockade of K(+) channels, since the toxin had no significant effect upon the K(+) current. In contrast, Lqh III inhibited the inactivation of a fraction of the Na(+) current, leading to a maintained late inward Na(+) current which represented about 45% of the peak Na(+) current, as observed during long-lasting depolarisations and in steady-state Na(+) current inactivation-voltage relationships when the pre-pulse potential was more positive than about -30mV. The activation kinetics of the late Na(+) current were well described by a single exponential whose time constant was 8.53+/-0.78 ms (n=3). Finally, Lqh III slowed the time-course of the remaining peak Na(+) current inactivation by altering initial amplitudes (to time zero of depolarisation) and time constants of its fast and slow phases. No significant additional effect was detected during the action of the toxin. In conclusion, we propose that, in frog myelinated axons, the effects of Lqh III are those typically attributed to classical scorpion alpha-toxins.

Action Potentials↗
PubMed2000

Physical structure of the excitable membrane of unmyelinated axons: X-ray scattering study and electrophysiological properties of pike olfactory nerve.

The aim of this work was to elicit correlations between physical structure and physiological functions in excitable membranes. Freshly dissected pike olfactory nerves were studied by synchrotron radiation X-ray scattering experiments and their physiological properties were tested by electrophysiological techniques. The scattering spectra contained a sharply oriented equatorial component (i.e. normal to the nerve axis), and an isotropic background. After background subtraction, the equatorial component displayed a weak and fairly sharp spectrum of oriented microtubules, and a strong and diffuse band of almost the same shape and position as the band computed for an isolated myelin membrane. We ascribed this spectrum to the axonal membranes. Under the action of temperature and of two local anesthetics, the spectrum underwent a contraction (or expansion) in the s-direction, equivalent to the structure undergoing an expansion (or contraction) in the direction perpendicular to the plane of the membrane. The main observations were: (i) with increasing temperature, membrane thickness decreased with a thermal expansion coefficient equal to -0.97(+/-0.19) 10(-3) degrees C(-1). The polarity and amplitude of this coefficient are typical of lipid-containing systems with the hydrocarbon chains in a disordered conformation. The amplitude and propagation velocity of the compound action potentials were drastically and reversibly reduced by lowering the temperature from 20 degrees C to 5 degrees C. (ii) Exposing the nerve to two local anesthetics (tetracaine and dibucaine) had the effect of decreasing membrane thickness. Action potentials were fully inhibited by these anesthetics. (iii) Upon depolarization, induced by replacing NaCl with KCl in the outer medium, approximately 25 % of the membranes were found to associate by apposing their outer faces. Electrophysiological activity was reversibly impaired by the KCl treatment. (iv) No detectable structural effect was observed upon exposing the nerves to tetrodotoxin or veratridine. Electrophysiological activity was fully impaired by tetrodotoxin and partially impaired by veratridine. The main conclusions of this work are that axonal membranes yield highly informative X-ray scattering spectra, and that these spectra are sensitive to the functional state of the nerve. These results pave the way to further studies of more direct physiological significance.

Anesthetics, Local↗
PubMed2000

Ca(2+) and Na(+) contribute to the swelling of differentiated neuroblastoma cells induced by equinatoxin-II.

Equinatoxin-II (EqTx-II), a cytotoxic protein (mol.wt 20 kDa) isolated from the sea anemone Actinia equina, was found to consistently increase the three-dimensional projected area of differentiated neuroblastoma (NG108-15) cells provided Ca(2+) was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethionate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells preloaded with the Ca(2+) indicator fura-2/AM revealed that EqTx-II (100 nM) markedly increased the fluorescence (F(340)/F(380)) ratio indicating a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). The elevation of [Ca(2+)](i) exhibited two components that seem to be related to the kinetics of EqTx-II-induced Ca(2+) entry since pretreatment of cells with Ca(2+)-ATPase inhibitors (thapsigargin), Ca(2+) channel blockers (nifedipine and Gd(3+)) or prolonged exposure to a high K(+) (75 mM) medium did not alter EqTx-II-induced Ca(2+) signals. As far as we know, this is the first demonstration that EqTx-II causes swelling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca(2+)](i) and needs the presence of extracellular Na(+). It is suggested that EqTx-II has the ability to insert into the plasma membrane of neuroblastoma cells and to form pores altering the membrane permeability and the intracellular osmolality, inducing a marked influx of water into the cells.

Animals↗
PubMed2000

Thioesterification of 2-arylpropionic acids by recombinant acyl-coenzyme A synthetases (ACS1 and ACS2).

2-Arylpropionic acids are a class of frequently used nonsteroidal anti-inflammatory drugs exhibiting a potent inhibition of cyclooxygenase isoforms supported by the (+)S-enantiomer alone. Nevertheless, some of these compounds in the (-)R configuration may undergo extensive inversion of configuration to their antipode. The key molecular basis for this mechanism invokes the stereoselective formation of the coenzyme A (CoA) thioester of the 2-arylpropionic acid by long-chain acyl-CoA synthetases (ACSs). In this report, rat recombinant ACS1 and ACS2 enzymes, constitutively highly expressed in adult rat liver and brain, respectively, have been overproduced in Escherichia coli strains and purified to homogeneity to investigate the involvement of these enzymes in the thioesterification of fenoprofen and ibuprofen. Recombinant ACS1 efficiently catalyzed both nonsteroidal anti-inflammatory drugs with Michaelis-Menten parameters of K(M) = 1686 +/- 93 microM, V(max) = 353 +/- 45 nmol/min/mg protein for (-)R-ibuprofen and K(M) = 103 +/- 12 microM, V(max) = 267 +/- 10 nmol/min/mg protein for (-)R-fenoprofen, and exhibited a marked stereoselectivity in favor of the (-)R-enantiomer. Recombinant ACS2, a closely related sequence with ACS1, exhibited a lower enzymatic efficacy from 7- to 130-fold for (-)R-ibuprofen and (-)R-fenoprofen, respectively. On the basis of these findings and considering the level of tissue expression of the different long-chain ACSs, ACS1 appears to be the major enzyme involved in the first step of the chiral inversion of 2-arylpropionic acids. Nevertheless, the participation of other ACS isoforms of minor quantitative importance could not be excluded in the thioesterification of xenobiotics.

Coenzyme A Ligases↗
PubMed1999

Hyperosmolar D-mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons.

The effects of hyperosmolar D-mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50-120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM D-mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na(+) entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar D-mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.

Action Potentials↗
PubMed1999

Neurotoxins targetting receptor site 5 of voltage-dependent sodium channels increase the nodal volume of myelinated axons.

The effects of a C57 type ciguatoxin (CTX-3C) and two types of brevetoxins (PbTx-1 and PbTx-3), known to bind to receptor site 5 of the neuronal voltage-dependent Na+ channel-protein, were studied on the morphology of living frog myelinated axons using confocal laser scanning microscopy. During the action of CTX-3C, PbTx-1, and PbTx-3 (10-50 nM), a marked swelling of nodes of Ranvier was observed without apparent modification of internodal parts of axons. In all cases, toxin-induced nodal swelling attained a steady-state within 75-100 min that was well maintained during an additional 90-115 min. The nodal swelling was reversed by an external hyperosmotic solution containing 100 mM D-mannitol and could be completely prevented by blocking voltage-dependent Na+ channels with 1 microM tetrodotoxin. It is suggested that CTX-3C, PbTx-1, and PbTx-3 by activating Na+ channels cause a continuous Na+ entry into axons, increasing internal Na+ concentration. Such an increase directly or indirectly disturbs the osmotic equilibrium between intra- and extra-axonal media, resulting in an influx of water, which is responsible for the long-lasting nodal swelling. Similar results were previously reported with two C60 type ciguatoxins (CTX-1B and CTX-4B). Thus, it is concluded that the four types of toxins targetting receptor site 5 of neuronal voltage-dependent Na+ channels, not only enhance nerve membrane excitability but also, on a long-term basis, cause a marked increase in the axonal volume.

Animals↗
PubMed1999

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+-dependent mechanism.

A novel conotoxin was isolated and characterized from the venom of the fish-hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 x higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+-free medium supplemented with ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA). Blockade of voltage-dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX-sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage-gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage-gated sodium channels.

Action Potentials↗
PubMed1999

Human and rat liver UDP-glucuronosyltransferases are targets of ketoprofen acylglucuronide.

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti-inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [beta-32P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite.

Animals↗
PubMed1999

[Ciguatoxins and brevetoxins: dissection of the neurobiological actions].

This review focuses on the neurobiological actions of ciguatoxins and brevetoxins which are phycotoxins produced respectively by the dinoflagellates Gambierdiscus toxicus and Ptychodiscus brevis. These actions are illustrated in particular by the effects of the toxins on myelinated nerve fibres and on skeletal neuromuscular junctions of vertebrates. Ciguatoxins and brevetoxins, through different vectors, are responsible for human intoxications characterized mainly by neurological disturbances. The molecular target of these families of lipid-soluble cyclic polyethers is the voltage-gated sodium channel, a fundamental transmembrane protein involved in cellular excitability. The different toxins share a common binding site (the receptor-site 5) located on the alpha sub-unit of this neuronal transmembrane protein. Electrophysiological studies of the mode of action of ciguatoxins and brevetoxins identify these toxins as specific sodium channel activators. Indeed, during the action of these phycotoxins, sodium channels remain permanently opened, at the resting membrane potential, which produces a continuous entry of sodium ions in most excitable cells. Such a sodium entry has various consequences on sodium-dependent physiological mechanisms, consisting in a membrane depolarization which, in turn, causes spontaneous and/or repetitive action potential discharges and thereby increases membrane excitability. These neuronal discharges may be transient or continuous according to the preparation and the toxin tested. The increase in membrane excitability during the action of ciguatoxins and brevetoxins is responsible for the different effects exerted by these toxins on various chemical synapses and secretory cells. Another consequence of the continuous entry of sodium ions into cells was revealed using confocal laser scanning microscopy and vital staining of plasma membranes with the fluorescent dye FM1-43. These techniques made feasible the dynamic study of morphological alterations produced by ciguatoxins and brevetoxins on various cellular preparations in situ. Thus, it has been possible to bring to the fore that these phycotoxins cause a marked increase in the volume of nodes of Ranvier of myelinated nerve fibres, motor nerve terminals innervating skeletal muscle and perisynaptic non-myelinating Schwann cell somata. This increase could be reversed by hyperosmotic external solutions and completely prevented by the blockade of voltage-gated sodium channels. The mechanisms involved in the increase in cellular volume, during the action of ciguatoxins and brevetoxins, are discussed.

Animals↗
PubMed1999

[Conus venoms: a source of toxins which interact with membrane- potential-dependent sodium channels].

Marine snails of the genus Conus, as they are carnivorous predators, have a venom apparatus used to capture their prey. The toxins contained in the venoms of Conidae, called conotoxins, are of a particular high degree of diversity and represent powerful tools in the neuroscience field. Indeed, these toxins specifically bind with a high affinity to receptors and ionic channels. Therefore, they provide original pharmacological tools which receive increasing investigation both to identify and study some functions of the nervous systems and to characterize new types and closely related subtypes of receptors or ionic channels. The voltage-gated sodium channel, because of its fundamental role in cell membrane excitability, is the specific target of a large number of animal and vegetal toxins. Actually, at least seven toxin receptor sites have been identified on this channel-protein. These toxins, and in particular conotoxins, are used to precise the role of different types and/or closely related subtypes of sodium channels in the peripheral and central nervous systems. The focus of the present review is to summarize our current knowledge of the consequences of physiological interactions between different conotoxin families and sodium channels.

Action Potentials↗
PubMed1999

A review on conotoxins targeting ion channels and acetylcholine receptors of the vertebrate neuromuscular junction.

In this article we present an overview of some peptides extracted and purified from the venom of marine snails of the genus Conus. These active peptides named conotoxins can be used as research tools to target voltage-gated ion channels as well as ligand-gated receptors. Because of their relatively small size, conotoxins can be chemically synthesized and made widely available. In this review we focus on conotoxins that target voltage-sensitive sodium channels, voltage-dependent calcium channels and nicotinic acetylcholine receptors of the vertebrate neuromuscular junction. Emphasis is given on summarizing our current knowledge of their primary structure and their specific pharmacological actions at the pre- and the post-synaptic level of the neuromuscular junction. Evidence is presented for conotoxins that discriminate between pre- and post-synaptic voltage-gated sodium channels. Among these peptides, the mu-conotoxin family is well characterized by its ability to block selectively sodium channels in skeletal muscle fibres without affecting axonal and nerve terminal Na+ channels. Furthermore, new conotoxins like Conus consors toxin (CcTx) and conotoxin EVIA selectively target Na+ channels in axons and nerve terminals without affecting skeletal muscle fibres. omega-conotoxins known as highly potent and selective blockers of voltage-sensitive calcium channels have proven to be valuable in determining the roles of the various subtypes of channels involved in acetylcholine release from motor nerve endings. Finally, Conus peptides which act at muscle nicotinic acetylcholine receptors constitute the most extensive characterized family of conopeptides that exhibit sequence similarity, different structural motifs and surprising diversity in their competitive and non-competitive actions.

Animals↗
PubMed1998

The wheat proteins puroindoline-a and alpha1-purothionin induce nodal swelling in myelinated axons.

The effects of two basic cysteine-rich lipid-binding proteins isolated from wheat seedlings, puroindoline-a and alpha1-purothionin, were studied on single frog myelinated axons stained with the fluorescent dye FM1-43 using confocal laser scanning microscopy. During exposure to either puroindoline-a or alpha1-purothionin (10 and 100 microM) a marked swelling of nodes of Ranvier was observed, provided NaCl was present in the external solution. It is suggested that these proteins increase the internal osmolality by forming pores in the axonal membrane and induce water influx to compensate for such an increase. Moreover, in the presence of alpha1-purothionin (100 microM), the intensity of the axonal staining with FM1-43 was increased. It is the first time, to our knowledge, that basic proteins containing domains of a cysteine-rich repeated motif are reported to produce swelling and water movements across neuronal cell membranes.

Animals↗
PubMed1998

Identification of PTEN/MMAC1 alterations in uncultured melanomas and melanoma cell lines.

A novel tumor suppressor gene, PTEN/MMAC1, has been recently shown to be mutated in gliomas, breast, prostate, kidney cancers and melanomas. Loss-of-heterozygosity studies in melanoma have suggested the presence of at least one chromosome 10q locus lost early in tumor progression. In this study, we screened 45 melanoma cell lines and 17 paired uncultured metastatic melanoma and peripheral blood specimens for PTEN/ MMAC1 alterations using PCR-SSCP and direct sequencing. We found nine melanoma cell lines with homozygous deletions (five with intragenic loss) and four cell lines with mutations (one nonsense and one frameshift; two intronic); from among our uncultured melanoma specimens, we found one tumor with a somatic 17 bp duplication in exon 7 leading to a premature stop codon and one tumor with a possible homozygous deletion. Furthermore, we have identified a novel intragenic polymorphism within intron 4 of PTEN/MMAC1. Taken together, these data suggest that PTEN/MMAC1 may be a chromosome 10q tumor suppressor important in melanoma tumor formation or progression.

Chromosomes, Human, Pair 10↗